环介导等温扩增
尿
磁性纳米粒子
纳米颗粒
材料科学
等温过程
纳米技术
化学
物理
生物化学
DNA
热力学
作者
Rahul Roy,Gagandeep Singh,Ujjwal Ranjan Dahiya,M. B. Pandey,Immaculata Xess,Dinesh Kalyanasundaram
摘要
Abstract Background Mucormycosis is a rare disease with scarce diagnostic methods for early intervention. Available strategies employing direct microscopy using calcofluor white-KOH, culture, radiologic, and histopathologic testing often are time-intensive and demand intricate protocols. Nucleic Acid Amplification Test (NAAT) holds promise due to its high sensitivity combined with rapid detection. Loop-mediated isothermal amplification (LAMP) based detection offers an ultrasensitive technique that does not require complicated thermocyclers like in PCR, offering a straightforward means for improving diagnoses as a near-point-of-care test. Methods The study introduces a novel magnetic nanoparticle-based LAMP assay for carryover contaminant capture to reduce false positives. Solving the main drawback of LAMP-based diagnosis techniques. The assay targets the cotH gene, which is invariably specific to Mucorales. Results The assay was tested with various species of Mucorales, and the LODs for Rhizopus microsporus, Lichtheimia corymbifera, Rhizopus arrhizus, Rhizopus homothallicus and Cunninghamella bertholletiae were 1fg, 1fg, 0.1pg, 0.1pg, and 0.01 ng respectively. This was followed by a clinical blindfolded study using whole blood and urine samples from 30 patients diagnosed with Mucormycosis. The assay has a high degree of repeatability and had an overall sensitivity of > 83%. Conclusions Early Mucormycosis detection is crucial, as current lab tests from blood and urine lack sensitivity and take days for confirmation despite rapid progression and severe complications. Our developed technique enables the confirmation of Mucormycosis infection in less than 45 minutes, focusing specifically on the RT-LAMP process. Consequently, this research offers a viable technique for quickly identifying Mucormycosis from isolated DNA of blood and urine samples instead of invasive tissue samples.
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